JP2022547098A - Novel micropeptide HMMW and its application - Google Patents

Abstract

The present invention discloses a micropeptide HMMW with a brand new structure and its application belonging to the field of biomedical research and development. The micropeptide HMMW is encoded by a human lncRNA, the recombinant vector is constructed such that the target cells highly express the micropeptide HMMW, and is used for human head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma, gastric cancer and breast cancer. It can inhibit the growth, migration, and in vivo tumor growth of a variety of solid tumors, including , renal, and skin cancers, and has potential drug development value, important tumor detection and therapeutic value. .

Description

The present invention relates to the field of biomedical research and development, and more specifically to the application of novel micropeptide HMMW in tumor detection and therapy.

Advances in transcriptomics, proteomics, and bioinformatic analytical methods have enabled scientists to discover that previously defined non-coding RNA molecules (IncRNAs, circRNAs, miRNAs, etc.) are actually small open reading frames with coding capacity ( sORF, small Open Reading Frame, nucleotide sequence does not exceed 300 bp), and the polypeptides encoded by sORF (sORF-encoded peptides, SEPs) consist of micropeptides, exceeding 100 amino acids in length. no). In the 2015 journal Cell, scientists screened skeletal muscle-specific lncRNAs encoding the polypeptide Myoregulin (MLN) by bioinformatics and were able to identify MLN as a key regulator of skeletal muscle physiology. announced that In the past two years, there are not many reports on IncRNAs that can encode micropeptides in the world, and the research fields are mainly focused on muscle differentiation and skeletal muscle development. Therefore, the discovery and identification of new micropeptides encoded by lncRNAs and the search for new applications are of great importance for opening the coding door of non-coding RNAs.

Tumors are a class of malignant diseases that seriously endanger human life and health.Uncontrolled growth, invasion and metastasis are the malignant manifestations of tumors and are also the leading causes of treatment failure and death. Currently, the main clinical treatments for cancer patients are surgery and chemotherapy (chemotherapy/drug therapy). For most tumors, surgery is suitable for definitive treatment of early-stage, intermediate-stage, and localized tumors, and palliative treatment of advanced tumors; The problem is that it is large, difficult to operate in some areas, and ineffective for asymptomatic metastasis. For most tumors, chemotherapy is suitable for intermediate and advanced tumors, and as a systemic therapy, chemotherapy is effective against primary tumors, metastases, and asymptomatic metastases; are low, and while achieving therapeutic efficacy, varying degrees of toxicity and side effects often occur. In addition, cancer patients undergoing long-term chemotherapy may develop new malignancies due to immunosuppression and direct carcinogenesis.

Polypeptide drugs have the advantages of high specificity, low toxicity and side effects, clear mechanism of action, and no harm to normal cells, tissues and organs. It has been gradually developed in the treatment of related diseases, metabolic diseases and infectious diseases. At present, more than 80 kinds of polypeptide drugs have been launched in the world, with a total annual sales of more than 20 billion US dollars, and China's polypeptide drug market sales continue its rapid growth momentum, but its Most have become mimetic polypeptide drugs.

Currently, the research of micropeptides in the detection and treatment of malignant tumors such as head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma is still blank, and this patent is a novel micro-micropeptide in tumors through bioinformatics analysis and experimental validation. Discover peptides and explore their applications in tumor detection and therapy to provide new solutions for tumor detection and therapy.

A first object of the present invention is to provide a novel micropeptide HMMW and its amino acid sequence, the micropeptide HMMW being the first novel human endogenous polypeptide discovered.

A second object of the present invention is to provide a nucleotide sequence, said nucleotide sequence obtainable by encoding said micropeptide HMMW.

A third object of the present invention is to provide a recombinant vector containing a nucleotide sequence encoding the micropeptide HMMW.

A fourth object of the present invention is to apply the micropeptide HMMW or a nucleotide encoding said micropeptide HMMW to the preparation of tumor detection reagents and tumor therapeutic agents, specifically human head and neck cancer, thyroid cancer. Including detection and therapeutic effects in cancer, lung cancer, esophageal squamous cell, gastric cancer, breast cancer, renal cancer and skin cancer.

Attached Figure 1 is a pcDNA3.1 plasmid map overexpressing the micropeptide HMMW. Attached Fig. 2 shows the expression status of the target band detected by western blot. Attached Figure 3 shows the inhibitory effect of the micropeptide HMMW on the proliferation of human head and neck cancer SCC4 cells. Attached Figure 4 shows the inhibitory effect of the micropeptide HMMW on the proliferation of human thyroid cancer SW579 cells. Attached Figure 5 shows the inhibitory effect of the micropeptide HMMW on the proliferation of human lung cancer A549 cells. Attached FIG. 6 shows the inhibitory effect of the micropeptide HMMW on the proliferation of human esophageal squamous cell carcinoma TE13 cells. Attached FIG. 7 shows the inhibitory effect of the micropeptide HMMW on the proliferation of human gastric cancer MGC803 cells. Attached FIG. 8 shows the inhibitory effect of the micropeptide HMMW on the proliferation of human breast cancer MDA-MB-231 cells. Attached FIG. 9 shows the inhibitory effect of the micropeptide HMMW on the proliferation of human kidney cancer UOK262 cells. Attached FIG. 10 shows the inhibitory effect of the micropeptide HMMW on the proliferation of human skin cancer A431 cells. Attached Figure 11 is the inhibitory effect of the micropeptide HMMW on tumor growth of human head and neck cancer SCC4 cells in vivo. Attached Figure 12 is the inhibitory effect of the micropeptide HMMW on tumor growth of human thyroid carcinoma SW579 cells in vivo. Attached Figure 13 is the inhibitory effect of the micropeptide HMMW on tumor growth of human lung cancer A549 cells in vivo. Attached Figure 14 shows the inhibitory effect of the micropeptide HMMW on tumor growth of human esophageal squamous cell carcinoma TE13 cells in vivo. Attached Figure 15 is the inhibitory effect of the micropeptide HMMW on tumor growth of human gastric cancer MGC803 cells in vivo. Attached Figure 16 is the inhibitory effect of the micropeptide HMMW on tumor growth of human breast cancer MDA-MB-231 cells in vivo. Attached Figure 17 shows the inhibitory effect of the micropeptide HMMW on tumor growth of human renal carcinoma UOK262 cells in vivo. Attached Figure 18 is the inhibitory effect of the micropeptide HMMW on tumor growth of human skin cancer A431 cells in vivo. Attached FIG. 19 shows the detection of the expression level of the micropeptide HMMW in cancer tissue/normal tissue by qPCR.

The technical solutions adopted in the present invention are as follows.

Application of the novel micropeptide HMMW in the preparation of tumor detection reagents and/or tumor therapeutic agents, wherein the amino acid sequence of said micropeptide HMMW comprises the sequence as shown in SEQ ID NO.2.

The application of the novel micropeptide HMMW in the preparation of a reagent for detecting tumors or therapeutic agents for tumors, wherein the amino acid sequence of said micropeptide HMMW is at least 85% or more homologous to the amino acid sequence as per SEQ ID NO. 1 (micropeptide HMMW I) (the corresponding micropeptides have been named HMMW II and HMMW III, as per SEQ ID NO. 2 and SEQ ID NO. 3), or have 90% homology (SEQ ID NO. 4 and The corresponding micropeptides are named HMMW IV and HMMW V, as per SEQ ID NO. 5) or have 95% homology (as per SEQ ID NO. 6 and SEQ ID NO. 7, the corresponding The micropeptides are named HMMW VI and HMMW VII), or have 98% homology (as per SEQ ID NO. 8 and SEQ ID NO. 9, the corresponding micropeptides are named HMMW VIII and HMMW IX). ), and those sequences with homology to SEQ ID NO. 1 retain similar functions of inhibiting tumor cell growth, proliferation, invasion or migration, and may be used as tumor detection reagents or tumor therapeutic agents. Can be used for preparation.

As used in this patent, "homology" refers to the percentage of identical or similar sequences in the comparison of two or more amino acid sequences. Percent homology can be measured by electronic methods such as the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.), which can measure two or more sequences according to various methods such as the Cluster method (Higgins, D.G. and P.M.Sharp (1988) Gene 73:237-244) can be compared. The Cluster method arranges the sequences in each group into clusters by examining the distances between all pairs. Then assign each cluster in pairs or groups. The percent homology between two amino acid sequences, such as sequence A and sequence B, is calculated by the following formula.

(Number of residues matched between sequence A and sequence B×100)/(number of residues in sequence A−number of spacer residues in sequence A−number of spacer residues in sequence B).

Application of the novel micropeptide HMMW in the preparation of tumor detection reagents or tumor therapeutic agents, said novel micropeptide HMMW having an amino acid sequence as per SEQ ID NO.

Said nucleotide is either a or b or c:

(a) Nucleotides encoding the micropeptide HMMW comprising the amino acid sequence as per SEQ ID NO.1.

(b) a nucleotide encoding the micropeptide HMMW having at least 85% homology with the amino acid sequence as per SEQ ID NO.1;

(c) a nucleotide sequence encoding the amino acid of claim 3, specifically a nucleotide sequence according to SEQ ID NO. 10-NO. 18, where N is A/T/G/C; ing).

Said recombinant vector comprising any of said nucleotides in said recombinant vector.

Application of any of the foregoing nucleotides in the preparation of tumor detection reagents and/or tumor therapeutic agents.

Said reagent test kit for tumor detection, wherein said reagent test kit contains a specific primer pair for any of said nucleotide sequences.

Preferably, the sequences of said specific primer pair are as shown in SEQ ID NO.19 and SEQ ID NO.20.

The agent is the above-mentioned agent for treating tumors, which comprises at least any of the micropeptides HMMW or any of the nucleotides or the recombinant vector, and a pharmaceutically acceptable vector.

Preferably, said drug is a drug having functions such as (a1)-(a4), in which:
(a1) to inhibit tumor growth;
(a2) to inhibit the growth of tumor cells;
(a3) to suppress tumor cell invasion;
(a4) to inhibit tumor cell migration;

Preferably, said tumor comprises human head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma, stomach cancer, breast cancer, renal cancer or skin cancer.

The beneficial effects of the present invention compared with the prior art are as follows.

(1) The present invention provides a micropeptide HMMW having a novel amino acid sequence, and as a result of searching databases (BLAST, UniProt) and literature, no protein or polypeptide fragment having sequence homology with the micropeptide HMMW was found. I didn't.

The micropeptide HMMW is composed of 51 amino acids and plays an important role in the detection and treatment of tumors, including human head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma, gastric cancer, breast cancer, renal cancer, and It can be used as a tumor marker for tumors, including skin cancers, ie the HMMW micropeptide is low expressed in said tumors.

(2) The present invention provides a micropeptide HMMW with a brand new amino acid sequence, said micropeptide HMMW can be used in human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells in vivo. , can significantly inhibit the proliferation, migration and invasion of gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, kidney cancer UOK262 cells, and skin cancer A431 cells.

Said micropeptide HMMW was used in vivo for human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, renal cancer UOK262 cells, skin It can significantly suppress the primary growth of cancer A431 cells.

In addition, the micropeptide HMMW can be used as a detection and therapeutic agent for malignant tumors, greatly expanding the therapeutic spectrum of the micropeptides and providing new ideas for the development of drugs for malignant tumors.

Specific Embodiments The invention will now be described in detail with reference to specific embodiments.

Example 1
In this example, the coding ability of HMMW micropeptides is detected.

The flag-tagged cDNA (nucleotide sequence of which is shown in SEQ ID NO. 10) constructed in vitro is an overexpression vector pcDNA-HMMW, and the map of the blank plasmid pcDNA3.1 is shown in FIG. It is as follows. The plasmid was introduced into 293T cells by lipo3000 liposomal transfection reagent, the cells were harvested 48 hours after transfection, centrifuged, the supernatant was discarded, the pelleted cells were collected, and the pelleted cells were rinsed twice with PBS. and collect the pelleted cells by centrifugation and discarding the supernatant. RIPA lysate is added to the collected pelleted cells and lysed on ice for 20 minutes, followed by centrifugation at 12,000 g for 10 minutes to collect the supernatant. Then add 1X SDS loading buffer, mix evenly by pipetting, and boil for 5 minutes for denaturation. Total proteins were separated on a 10% SDS-PAGE gel, transferred to a PVDF membrane, blocked with 5% non-fat dry milk at room temperature for 2 hours, incubated with Flag primary antibody (abeam) overnight at 4°C, and washed with TBST. Wash 3 times. Secondary antibody is incubated for 1 hour at room temperature and washed 3 times with TBST. Develop with ECL ultrasensitive chemiluminescent solution and detect the presence or absence of the target band with a Tannon imaging system.

The results are shown in Figure 2 and Western blotting detected the appearance of the target band of the micropeptide HMMW after transfection of the recombinant plasmid, demonstrating that the micropeptide HMMW protein has the ability to encode. shows further.

Example 2
In this example, the micropeptide HMMW I-IX was obtained by solid phase synthesis and the resulting micropeptide HMMWI-IX is checked.

Synthesizing the micropeptide HMMWI-IX (whose amino acid sequence is as shown in SEQ ID NO. 1-9) by a solid-phase polypeptide synthesis method, and separating and purifying the synthesized micropeptide HMMW by preparative HPLC, Purity of the micropeptide HMMW is determined by analytical RP-HPLC. Polypeptide solid-phase synthesis method uses Fmoc-wang-resin or Fmoc-CTC-resin as a starting material, sequentially binds dipeptides to 51 peptides with protected amino acids, and after the completion of peptide binding, thoroughly wash the peptides. Cleavage and post-processing yield the crude product of the angiogenesis inhibitor. The crude product is dissolved and purified twice by preparative high performance liquid phase, concentrated and lyophilized to give the pure product and finally a third purification gives the micropeptide purified product. This method can not only guarantee the efficiency of the synthesis, but also improve the purity of the product. Details are as follows.

1. Conjugation of peptides (including conjugation from dipeptides to 51 peptides):
Weigh an appropriate amount of Fmoc-wang-resin or Fmoc-CTC-resin, pour it into a glass sand core reaction column, add an appropriate amount of CH2Cl2 to make the resin fully swell.

a. Decap: add an appropriate amount of hexahydropyridine/N,N-dimethylformamide (DMF) decap solution, react for a period of time, then suck out the decap solution completely, wash once with DMF in the middle, then repeat An appropriate amount of decap solution is added and allowed to react to remove the Fmoc protecting group.

b. Wash: Aspirate the decap solution completely and wash the resin with DMF to thoroughly wash away the by-products.

c. Condensation: The protected amino acid and activator for peptide binding are dissolved in DMF and condensing agent, shaken well, and the temperature is controlled at about 34°C to allow complete reaction in the reactor.

d. Wash: Aspirate the reaction solution thoroughly and wash the resin thoroughly with DMF to wash away the by-products.

2. Peptide Cleavage:
Put the completely aspirated resin into a round-bottomed flask, add the 36 peptide intermediates completely dissolved in the cleavage solution, separate the resin from the polypeptide by a sand core funnel, and determine the components of the cleavage solution and the volume of each component. The composition is trifluoroacetic acid: phenol: water: thioanisole: EDT = 90:3:3:2:2.

3. Post-processing:
Anhydrous ether is first added to the cleaving solution to separate the polypeptide, followed by centrifugation, discarding the supernatant, washing the polypeptide with anhydrous ether, and aspirating off completely before extracting the crude polypeptide. obtain.

4. Purification:
a. Dissolution: Weigh the crude product to prepare a 5-20 g/L solution and filter through a mixed filter membrane with a pore size of 0.45 μm.

Preparation: Single, double, and triple purification by semi-preparative high-performance liquid chromatography to obtain qualified products of purified polypeptide Mobile phase: A is acetonitrile, B is 0.1% TFA in water It has become.

Purify once: Rinse the equilibrated preparative column with 10%-90% acetonitrile and 20%-80% buffer at a flow rate of 50 mL/min-100 mL/min for 10 minutes. The dissolved and filtered crude product is sample loaded with an infusion pump.

Solutions with absorbance values greater than 200 mV at UV wavelength 220 nm are collected and those >95% pure are detected and combined as peak tops for secondary separation and purification.

Double Purification: After removing the organic solvent by rotary evaporation of the peak tops from the first purification, 5-95% acetonitrile and 15-85% buffer at a flow rate of 50-100 mL/min. , load the sample with the infusion pump.

Solutions with absorption values greater than 200 mV at UV wavelength 220 nm are collected and those with greater than 98% purity are detected and qualified.

b. Concentration, filtration, and lyophilization: Concentrate the qualified solution under vacuum on a rotary evaporator at 37°C to remove residual solvent and water for injection. Finally, it is filtered through a 0.22 μm filter membrane, and the filtrate is placed in freeze-drying trays and freeze-dried in a freeze-dryer to obtain a pure product.

Triplicate Purification: Qualified samples >98% pure from duplicate purifications were purified in triplicate with 5-95% acetonitrile and 10-90% buffer at flow rates of 50-100 mL/min. , to prepare a purified product of the polypeptide.

More than 99.5% of samples collected and detectably combined with absorbance values greater than 200 mV at UV wavelength 220 nm result in purified qualifying product.

5. Purity Detection The purified product is collected after lyophilization and the purity of the polypeptide is detected by analytical RP-HPLC. The analytical conditions were mobile phase: ACN (+0.1% TFA), H2O (+0.1% TFA); acetonitrile linear gradient: 10%-100%; flow rate: 1 mL/min; run time: 20 min; sample loading volume: 20 μL. ; Detection wavelength: 220 nm.

The purity of the synthesized micropeptides was assessed by reversed-phase liquid chromatography analysis, which indicated that the nine prepared micropeptides HMMW were all over 95% pure, meeting the design requirements.

In this experiment, the micropeptide HMMW I-IX was successfully synthesized by solid-phase synthesis, which makes the method highly reproducible, easy to handle, and less contaminating. In experiments, polypeptides can be synthesized with two types of resin (wang resin or CTC resin). Experimental wang resin is relatively stable compared to other resins, with fewer side reactions, better crude peak shape, and higher relative purification yields, resulting in relatively lower costs. there is The reaction of the experimental CTC resin is less affected by temperature and has a faster reaction rate. Alternatively, the polypeptide is purified by reversed phase high performance liquid chromatography. Compared to isocratic elution, gradient elution has better separation efficiency, suitable retention time during separation, higher production efficiency and higher purity.

Example 3
This example detects the effect of the micropeptide HMMWI-IX on the proliferative capacity of human tumor cells.

Human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, kidney cancer UOK262 cells, skin cancer A431 cells were incubated at 37°C at 5%. Harvested by trypsin when cultured to 90% confluency in CO2 incubation, cells resuspended in medium, counted microscopically, adjusted cell concentration to 3.0×10 ⁴ cells/mL and placed in cell suspension. The suspension is inoculated into 96-well plates, 100 μL per well, and cultured overnight in a 37° C., 5% CO 2 incubator. After the cells were completely attached to the wall, different doses of the micropeptide HMMW I-IX were administered as the administration group, Taxol as the positive control group, and drug-free medium as the blank control group. Dilute each to a predetermined concentration. Add 100 μL of each dilution to a 96-well plate and incubate in an incubator at 37° C., 5% CO 2 for 48 hours. Add 20 μL of 5 mg/mL MTT to each well of the 96-well plate and continue to culture for 4 hours. Aspirate the medium and add 100 μL of DMSO to each well to dissolve. Absorbance at a detection wavelength of 570 nm and a reference wavelength of 630 nm was measured using a microplate reader to calculate the growth inhibition rate (PI). The formula is PI (%) = 1 - treatment group/negative group. The experiment was repeated three times independently, the results obtained by the experiment are shown as mean±SD, and a statistical T-test was performed, *P<0.05 for significant difference, **P<0.01 for highly significant difference. It has become.

The results are shown in Figures 3-10, and compared with the negative control group, at doses of 1-32 μM, micropeptide HMMW-I inhibited human head and neck cancer SCC4 cells (Figure 3), thyroid cancer SW579 cells (Figure 4), Lung cancer A549 cells (Fig. 5), esophageal squamous cell carcinoma TE13 cells (Fig. 6), gastric cancer MGC803 cells (Fig. 7), breast cancer MDA-MB-231 cells (Fig. 8), kidney cancer UOK262 cells (Fig. 9), skin cancer The proliferation of A431 (Fig. 10) could be significantly inhibited to different extents, indicating a dose-dependent relationship. The results are also shown in Table 5, and compared with the negative control group, both the micropeptide HMMW I-IX and human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, and esophageal squamous cells at doses of 1-32 μM. It can significantly suppress the proliferation of epithelial cancer TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, kidney cancer UOK262 cells, and skin cancer A431 cells, indicating a dose-dependent relationship. All of the polypeptides with more than 85% homology to the original sequence HMMW I have been shown to be effective in suppressing tumor cell growth, and the micropeptide HMMWI-IX can be considered as a candidate anti-tumor drug. .

Example 4
Effect of micropeptide HMMW I-IX on migration ability of human tumor cells Human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, Renal carcinoma UOK262 cells, skin carcinoma A431 cells are seeded in transwell chambers and 100 μL per well and different doses of the micropeptide HMMWI-IX are added to each chamber. Next, 0.6 mL of complete medium containing 10% FBS is added to the lower chamber of the transwell to stimulate cell migration and cultured at 37°C, 5% CO2 for 24 hours. Discard the medium in the wells, fix with 90% alcohol at room temperature for 30 minutes, stain with 0.1% crystal violet at room temperature for 10 minutes, rinse with clean water, gently wipe off non-migrated cells on the upper layer with a cotton swab, Observe under a microscope, select four fields of view, take pictures, and count them. Calculate the migration inhibition rate (MIR) of cells according to the following formula.

where N _test is the number of cells migrated in the test group (1, 4, and 14 µM dose groups in the table), and N _control is the number of cell migration in the blank control group (0 µM dose groups in the table). It has become.

The experiment was independently repeated three times, the results obtained by the experiment were calculated as the mean ± SD, and a statistical t-test was performed, where the experiment was independently repeated three times, as shown in the table. After repeating the experiment three times for each dose of any type of cells, the number of cell migration (Mean±SD) can be calculated according to the above formula. P values represent statistically significant differences, and statistical significance of results provides a way of estimating the true degree (representative of the whole) of the results, *P<0.05 being a significant difference, **P<0.01 The difference is very significant.

The results are shown in Table 6, and both the micropeptide HMMW I-IX and human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, and esophageal squamous cell carcinoma at doses of 1-16 μM compared to the negative control group. The migration of TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, renal cancer UOK262 cells, and skin cancer A431 cells can be significantly suppressed at different degrees, indicating a dose-dependent relationship. All polypeptides with more than 85% homology to the original sequence HMMW I are effective in inhibiting tumor cell migration, indicating that they can be used as therapeutic agents to inhibit the migratory capacity of malignant tumor cells.

Example 5
Effect of the micropeptide HMMW I-IX on the invasive capacity of human tumor cells.
10 mg/mL Matrigel is diluted 1:3 with medium, applied to transwell chamber membranes and allowed to air dry at room temperature. Human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, kidney cancer UOK262 cells, skin cancer A431 cells cultured to log phase Cells are digested with trypsin, harvested, washed twice with PBS, and resuspended in blank medium. Adjust the cell concentration to ¹ x 105 cells/mL. Different doses of the micropeptide HMMW I-IX are added to each chamber while cells are seeded in transwell chambers at 100 μL per well. Add 0.6 mL of complete medium containing 10% FBS to the lower chamber of the transwell to stimulate cell invasion and incubate at 37°C, 5% CO2 for 24 hours. Discard the medium in the wells, fix with 90% alcohol at room temperature for 30 minutes, stain with 0.1% crystal violet at room temperature for 10 minutes, rinse with clean water, gently wipe off non-infiltrated cells on the upper layer with a cotton swab, Observe under a microscope, select four fields of view, take pictures, and count them. Calculate the invasion inhibition rate (IIR) according to the following formula.

Here, N _test is the number of cell infiltration in the test group (1, 4, and 16 μM dose groups in the table), and N _control is the number of cell infiltration in the blank control group (0 μM dose group in the table). It has become. Experiments are repeated three times independently, experimental results are calculated as mean±SD, and statistical t-tests are performed. P values represent statistically significant differences, and statistical significance of results provides a way of estimating the true degree (representative of the whole) of the results, *P<0.05 being a significant difference, **P<0.01 The difference is very significant.

The results are shown in Table 7, and both the micropeptide HMMW I-IX and human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, It can significantly suppress the migration of kidney cancer UOK262 cells and skin cancer A431 cells to different extents, indicating a dose-dependent relationship. All of the polypeptides with more than 85% homology to the original sequence HMMW I have the effect of suppressing tumor cell invasion and can be used as therapeutic agents to suppress the invasion ability of malignant tumor cells. there is

Example 6
Effect of the micropeptide HMMW on human tumor cell growth in vivo.
(1) Human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, renal cancer UOK262 cells were cultured in large amounts and 0.25% Digestion with trypsin solution, after completion of digestion, cell suspension was centrifuged at 1000 rpm for 5 minutes, cells were resuspended in serum-free DMEM medium, counted, and cell concentration was adjusted to 5×10 ⁷ cells/ml. adjust.

(2) Group-specific cell suspensions corresponding to the left armpit of each nude mouse (purchased female mice weighing 14-16 g, 4-6 weeks old, and adapted for 1 week in an SPF class animal breeding room) 100 μl of the liquid was inoculated, and the injection amount of cells was 5×10 ⁶ cells.

(3) After inoculation, the nude mice were carefully observed for tumor growth at the inoculation site. Seven days after inoculation, white nodules appeared at the inoculation site. A hard tumor mass gradually formed at the site, and the average volume of tumor tissue reached 100 mm3 in about 14 ^days . 15 mg/kg micropeptide HMMW dose is divided into a low dose group and 15 mg/kg micropeptide HMMW dose is a high dose group), each group has 6 animals, and the animals weighed between 16 and 16 at the start of dosing. It is 18g.

(4) The volume of the implanted tumor was measured and recorded every 2 days, and the formula for calculating tumor volume (TV) is as follows.
Tumor volume = 0.5 × a × b^2
Here, a is the length (mm) of the transplanted tumor, and b is the width (mm) of the transplanted tumor.

The results are shown in Tables 11-18, and compared with the saline control group, the micropeptide HMMW increased human head and neck cancer SCC4 cells (Fig. 11), thyroid cancer SW579 cells (Fig. 12), lung cancer A549 cells (Fig. 13). ), esophageal squamous cell carcinoma TE13 cells (Fig. 14), gastric cancer MGC803 cells (Fig. 15), breast cancer MDA-MB-231 cells (Fig. 16), kidney cancer UOK262 cells (Fig. 17), skin cancer A431 cells (Fig. 18) The in vivo tumorigenic potential of HMMW can be significantly suppressed to different extents, demonstrating a dose-dependent relationship, and the micropeptide HMMW can be regarded as a novel anti-tumor polypeptide.

Example 7
Effect of the micropeptide HMMW I-IX on human tumor cell growth in vivo.
(1) Human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells, gastric cancer MGC803 cells, breast cancer MDA-MB-231 cells, renal cancer UOK262 cells were cultured in large amounts and 0.25% Digestion with trypsin solution, after completion of digestion, cell suspension was centrifuged at 1000 rpm for 5 minutes, cells were resuspended in serum-free DMEM medium, counted, and cell concentration was adjusted to 5×10 ⁷ cells/ml. adjust.

(2) Group-specific cell suspensions corresponding to the left armpit of each nude mouse (purchased female mice weighing 14-16 g, 4-6 weeks old, and adapted for 1 week in an SPF class animal breeding room) 100 μl of the liquid was inoculated, and the injection amount of cells was 5×10 ⁶ cells.

(3) After inoculation, the nude mice were carefully observed for tumor growth at the inoculation site. Seven days after inoculation, white nodules appeared at the inoculation site. A hard tumor mass gradually formed at the site, and the average volume of tumor tissue reached 100 mm3 in about 14 ^days . I-IX were divided into one group, each with a dose of 15 mg/kg), each group had 6 animals, and the animals weighed 16-18 g at the start of administration.

(4) The volume of the implanted tumor was measured and recorded every 2 days, and the formula for calculating tumor volume (TV) is as follows.
Tumor volume = 0.5 × a × b^2
Here, a is the length (mm) of the transplanted tumor, and b is the width (mm) of the transplanted tumor.

The results are shown in Table 8, and compared with the saline control group, the micropeptide HMMW I-IX reduced human head and neck cancer SCC4 cells, thyroid cancer SW579 cells, lung cancer A549 cells, esophageal squamous cell carcinoma TE13 cells, and gastric cancer MGC803 cells. It can significantly inhibit the in vivo tumorigenicity of cells, breast cancer MDA-MB-231 cells, kidney cancer UOK262 cells, and skin cancer A431 cells, indicating a dose-dependent relationship. The micropeptide HMMW I-IX can be regarded as a novel anti-tumor polypeptide, since all polypeptides with more than 85% homology with the original sequence HMMW I are effective in inhibiting tumor cell growth.

Example 8
Expression of HMMW in tumor patients and normal paracancerous tissues.
Head and neck cancer, brain glioma, thyroid cancer, esophageal squamous cell carcinoma, lung cancer, liver cancer, stomach cancer, kidney cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, colorectal cancer, pancreatic cancer, bone cancer by TCGA standard method We downloaded the RNA-seq sequence files and clinical information of cancer and normal tissues from 16 types of tumors, including sarcoma and skin cancer, and analyzed differential expression of the micropeptide HMMW (criteria: (1) | cancer/paracancerous). Analyze the expression level|>2, (2) P<0.05).

As shown in Table 9, the expression levels of the micropeptide HMMW in eight tumor tissues of human head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma, gastric cancer, breast cancer, renal cancer and skin cancer compared with normal tissues. is significantly reduced. We showed that the expression of the micropeptide HMMW was significantly negatively correlated with the development of various tumors.

Example 9
Expression of HMMW in clinical patients with head and neck cancer and normal paracancerous tissues.
(1) Specimen Collection With informed consent of the patient, specimens of head and neck cancer and paracancerous tissue were collected during surgery and washed with saline followed by liquid nitrogen or - Store in a refrigerator at 80°C.

(2) Primer design

Primers were designed with Primer Premier 5.0 according to the nucleotide sequence corresponding to the micropeptide HMMW, and the sequences are as follows.
Upstream primer (sequence shown in SEQ ID NO. 3)
Downstream primer (sequence shown in SEQ ID NO. 4)

(3) Detect the expression of HMMW in head and neck cancer patients and normal paracancerous tissues by real-time quantitative PCR.

Extract total RNA of the collected samples according to life's Trizol instructions, then quantify the purity and concentration of the extracted RNA with a NanoDrop ND-1000 Nucleic Acid Quantitator, and then quantify the extracted RNA with an agarose quality check. ensure the integrity of The extracted total RNA is reverse transcribed into cDNA using the TaKaRa reagent test kit PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time). qPCR reactions are performed with the TaKaRa Reagent Test Kit SYBR® Premix Ex Taq ^™ II (TliRNaseH Plus). The reaction system is shown in the table below.

After uniformly mixing the above components, the following procedure is followed: predenaturation at 95°C for 30s, 40 cycles, 95°C for 5s, 60°C for 30s. The specificity of the reaction is judged according to the melting curve and the relative expression level of HMMW is calculated by the 2- ^ΔΔCt method. The results are shown in Figure 19 and show that in about 75% of head and neck cancer samples, HMMW expression levels are significantly reduced compared to normal paracancerous tissue.

Claims (10)

Amino acid sequence of the micropeptide HMMW is an amino acid sequence having at least 85% homology with the amino acid sequence as shown in SEQ ID NO. 1. Application of micropeptide HMMW. Application of said novel micropeptide HMMW in the preparation of tumor detection reagents or tumor therapeutic agents according to claim 1, characterized in that the amino acid sequence of the micropeptide HMMW comprises the amino acid sequence as per SEQ ID NO.1. 2. The novel micropeptide HMMW in the preparation of a tumor detection reagent or tumor therapeutic agent according to claim 1, wherein the novel micropeptide HMMW has any one of the amino acid sequences shown in SEQ ID NOs. 1 to 9. Application of micropeptide HMMW. The nucleotide, wherein the nucleotide is any one of (a) or (b) or (c).
(a) a nucleotide sequence encoding said amino acid sequence comprising SEQ ID NO.2;
(b) a nucleotide encoding the micropeptide HMMW of claim 2;
(c) a nucleotide sequence encoding the amino acid of claim 3, specifically a nucleotide sequence as per SEQ ID NO. 10 to NO. 18, where N is any of A/T/G/C; there is Said recombinant vector, characterized in that said recombinant vector comprises a nucleotide according to claim 4. Application of a nucleotide according to claim 4 or a recombinant vector according to claim 5 in the preparation of a tumor detection reagent or tumor therapeutic. The reagent test kit for tumor detection, wherein the reagent test kit comprises a specific primer pair designed by the nucleotide sequence of claim 4. 8. The tumor detection reagent test kit according to claim 7, wherein the specific primer pair is as shown in SEQ ID NO.19 and SEQ ID NO.20. 8. The tumor detection reagent test kit according to claim 7, wherein the tumor includes human head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma, stomach cancer, breast cancer, renal cancer, and skin cancer. A pharmaceutical composition for tumor therapy, characterized in that it comprises the micropeptide HMMW comprising at least the amino acid sequence of SEQ ID NO. 1, or the nucleotide according to claim 4, or the recombinant vector according to claim 5. The pharmaceutical composition of

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